The EGF receptor is the first member of the ErbB family of growth factor receptor tyrosine kinases that also includes ErbB2, ErbB3 and ErbB4. Binding of EGF to its receptor induces the formation of EGF receptor homodimers, resulting in autophosphorylation of the EGF receptor. The EGF receptor is able to heterodimerize with other members of the ErbB family, with ErbB2 being the preferred heterodimerization partner. Recent data suggest that ErbB receptors may form heterotetramers and that activated oligomers may dissociate into monomers that re-dimerize with inactive monomers in a process referred to as secondary dimer formation. EGF shows heterogeneity in affinity when binding to its receptor. This observation has been attributed to the existence of two independent classes of sites. How these arise from the expression of a single EGF receptor protein has never been explained. Recently, we have shown that EGF binding can be described by a model that involves negative cooperativity in an aggregating system. In this model, the affinity of EGF for a monomer and an unoccupied dimer is similar. However, binding to the second site on an EGF receptor dimer is negatively cooperative. This model predicts that the negative cooperativity will lead to EGF-induced dissociation of the dimer at high concentrations of EGF, a prediction that we have confirmed using fluorescence correlation spectroscopy and enzyme complementation to examine EGF receptor oligomerization. Our long-term goal is to understand how ErbB receptors are activated and to develop a model for predicting the interactions among ErbB family members. Using our new model as the starting point for further investigations, the specific aims of this proposal are to: 1) Quantify the interaction of the EGF receptor and ErbB2 through ligand binding studies;2) Determine whether EGF receptor-mediated activation of ErbB2 occurs via the formation of a hetero-tetrameric complex and/or involves dissociation of dimeric receptor complexes;and, 3) Determine whether different EGF receptor ligands induce characteristic patterns of EGF receptor oligomers and whether this can be related to differences in downstream signaling. EGF equilibrium binding studies will be used to quantitatively characterize the interaction of the EGF receptor with ErbB2. Fluorescence correlation spectroscopy and brightness analysis along with enzyme complementation will be employed to assess the oligomerization state of EGFR/ErbB2 heteromeric complexes while chemically-induced dimer formation will be used to probe the activation mechanism of ErbB2 within these complexes. A combination of these methods will be applied to assess oligomerization of the EGF receptor elicited by three different agonists. Signaling stimulated by the three agonists will be assessed by measuring the activation of downstream pathways. Together, these data will characterize the interactions of the EGF receptor and ErbB2 allowing the development of a quantitative model for the activation of ErbB2 via the EGF receptor and provide insight into the role of ErbB family oligomers in the process of cell signaling.